Project Summary The RNA genomes of important human pathogens such as poliovirus, heptatitis C and ebola virus are replicated by virally encoded RNA-dependent RNA polymerases (RdRp), an establish anti-viral target. The molecular mechanisms of RdRp function will only be understood once we have both characterized its accessible structural states and delineated the transitions between these states as RdRp progresses through its catalytic cycle. This information would be critical for predicting fidelity-altering mutations that alter this process, and/or for designing small molecule modulators of RdRp function that would interfere with the structural transitions. Crystal structures, by themselves, have been unable to capture the full range of structural rearrangements necessary for RdRp function, and give no information about the timescale of the conformational fluctuations. For example, active-site remote mutations that change RdRp fidelity and virus biology, do not lead to substantial structural differences, but rather, they change RdRp protein fluctuations over multiple timescales. In this grant application, we propose to use solution-state nuclear magnetic resonance (NMR) to watch the conformational rearrangements in an archetypal RdRp (in our case, from poliovirus) throughout its nucleotide addition cycle, contrast these conformational dynamics between wild-type and low/high fidelity-mutant RdRps, and delineate the molecular mechanisms of a novel class of nucleoside analogs, which include members under clinical trials. We predict that the fidelity-mutations and the nature of the incoming nucleotide ('correct' or 'incorrect' Watson-Crick base-pair) will change the kinetics and/or thermodynamics of structural rearrangements critical for RdRp function. We also propose that the fidelity- altering, remote-site mutations exert their effects through a long-range, amino acid network. We will delineate this network through kinetic and NMR studies of selected mutants, including mutants derived from the Sabin 1 vaccine strain (i.e. a clinically-used, orally bioavailable vaccine strain for poliovirus). We predict that RdRp mutations contribute to the Sabin attenuated phenotype through altering RdRp fidelity. Understanding the interactions for coordinating the structural rearrangements in RdRp will allow us to predict amino acid changes that interfere with these motions and alter polymerase function. Such mutations in RdRp would be predicted to change polymerase fidelity, and therefore may serve as the basis of novel vaccine strains. Small molecules may also be used to perturb the structural rearrangements in RdRps; our studies will serve as a basis for illuminating the poorly understand mechanisms of actions for these compounds. Structure and dynamics are highly conserved among RdRps, so these concepts will be applicable to RNA viruses in general.